Protein chip is useful for analysis of a protein-binding molecule and the like. To attain such objects, protein should be stably immobilized in the same orientation. An entrapment method (Journal of Electroanalytical Chemistry, vol. 347, pp 293-301 (1993)) and a polymerization method (Journal of Electroanalytical Chemistry, vol. 511, pp 128-133 (2001)) have been known as typical immobilizing techniques for proteins. However, either of those methods has a problem in that the orientation of immobilized proteins cannot be controlled. Self-accumulation method (Sensors and Actuator B, vol. 24-25, pp 113-116 (1995)) is also known that utilizes mercapto-bond of a sulfur atom of cysteine to the surface of a metal such as gold having high crystallinity. However, the self-accumulation method has problems such as a decrease of activity of a protein and disarray of orientation because cysteine residues exist in many parts of the protein structure and reaction proceeds in each of the cysteine residues. In addition, the immobilization methods as described above are irreversible, which does not allow immobilized proteins to re-dissociate, so that it is difficult to reuse substrates and proteins, and also difficult to analyze a small amount of samples bound to the immobilized proteins.
Another technique in which proteins are bound to metal ions by using a metal-binding polypeptide such as polyhistidine fused to a protein in order to control the orientation of immobilized proteins is also known (JP 2001-083155 A). However, a bond between the metal-binding polypeptide and the metal ion is apt to dissociate because of a competitive reaction of coordination bond, and therefore the method is not suitable for stable immobilization.